Throughout this application various publications are referred to by author and year within brackets. The full references are listed alphabetically after the Experimental Section. The disclosures for these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Ribozymes are RNA molecules that can cut or ligate other nucleic-acid molecules (usually RNA) in a catalytic fashion (Cech and Bass, 1986; Altman, et al., 1987). The hammerhead ribozyme is one of the best-known ribozymes. It has been studied extensively in isolated chemical systems (Forster and Symons, 1987; Uhlenbeck, 1987; Haseloff and Gerlach, 1988; Jeffries and Symons, 1989; Koizumi, et al., 1988), and used in gene-control studies in living cells (Cotten and Birnstiel, 1989; Cameron and Jennings, 1989; Sarver, et al., 1990; Saxena and Ackerman, 1990; Sioud and Drlica, 1991; Sioud, et al., 1992). A hammerhead ribozyme as defined by Haseloff and Gerlach (Haseloff and Gerlach, 1988) is shown in FIG. 1. It contains two stretches of conserved nucleotides (boxed), a stem-loop structure (bases 18-29) containing helix II, and flanking nucleotides which form double-helices I and III in combination with the substrate.
The instability of ribozymes in living cells is a major concern. One approach taken to protect transcribed ribozymes from nuclease attack in cells has been to embed the ribozyme in a larger, folded structure. Thus, hammerhead ribozymes have been placed next to the anti-codon loop in t-RNA.sup.met (Cotten and Birnstiel, 1989), the 3' untranslated region of the luciferase gene (Cameron and Jennings, 1989), and in a molecule with bacteriophage T7 transcription terminator at its 3' end (Sioud, et al, 1992). These ribozymes appeared to be more stable than the corresponding, unprotected ribozymes; however, in the only comparative study, the stabilized ribozyme did not cleave more target RNA than the shorter-lived ribozyme, indicating that the protecting structure may decrease the specific activity of that ribozyme (Sioud, et al., 1992).
An alternative approach has been to chemically synthesize ribozymes with ribonucleotides modified at the 2' position. The modified nucleotides have included 2'-deoxy- 2'fluoro-, 2'-amino-, 2'-O-allyl- and 2'-O-methyl-ribonucleotides (Perreault, et al., 1990; Perreault, et al., 1991; Olsen, et al., 1991; Pieken, et al., 1991; Williams, et al., 1992; Paolella, et al., 1992). A ribozyme consisting predominantly of 2'-O-allyl ribonucleotides displayed greatly improved stability compared to an unmodified ribozyme in the presence of bovine serum (Paolella, et al., 1992). Modifications to nucleotides in the hybridizing arms and/or in helix II of the ribozyme have little effect on catalytic efficiency (Olsen, et al., 1991; Pieken, et al., 1991; Williams, et al., 1992; Paolella, et al., 1992); for example, substitution of the 2'-hydroxyl groups with 2'-O-allyl groups in all non-conserved nucleotides of a hammerhead ribozyme resulted in full retention of activity (Paolella, et al., 1992). On the other hand, changing the 2'-substituent in any of the conserved nucleotides of the ribozyme resulted in a decrease in catalytic activity, the magnitude of which varied greatly depending on the number of changes, the nature of the change, and the particular nucleotides modified (Perreault, et al., 1990; Perreault, et al., 1991; Olsen, et al., 1991; Pieken, et al., 1991; Williams, et al., 1992; Paolella, et al., 1992).